What's the first step in determining a protein sequence?
What can proteins be seperated based on?
- binding ability
How are proteins isolated from an assay?
A seperation technique that seperates molecules based on size using a semipermeable membrane e.g. cellulose
Describe gel-filtration chromatography
A seperation technique that seperates based on size. The sample is applied to the top of the column consisting of porous beads. The large molecules flow more rapidly through the column.
Describe ion-exchange chromatography
A separation technique that separates based on net-charges.
If a protein has a net positive charge (at pH7), it will bind to a column of beads containing carboxylate groups, whereas a negatively charged protein will not.
The bound protein can then be eluted.
Describe affinity chromatography
A separation technique that separates based on the chemical groups present.
This technique takes advantages of the high affinity of many proteins for specific chemical groups.
Good for isolating transcription factors.
Describe high-pressure liquid (HPLC) chromatography
- Is an enhanced version of column separation
- Column materials more refined
- Increased resolution and flow rate
What is the next stage in separation after the purification of proteins?
Separation of the purified proteins by gel electrophoresis
The principle of ___ _____________ is that a molecule with a net charge will move in an electric field. The velocity of _________ (v) of a protein in an electric field depends on the ________ _______ _________ (E), the ___ _______ of the protein (z), and the __________ ___________ (f).
The principle of gel electrophoresis is that a molecule with a net charge will move in an electric field. The velocity of migration (v) of a protein in an electric field depends on the electric field strength (E), the net charge of the protein (z), and the frictional coefficient (f).
V = Ez/f
Velocity of migration of a protein = (electric field strength X net charge of protein) / frictional coefficient
What does the frictional coefficient of a protein depend upon?
The mass and shape of the migrating molecule, and the viscocity of the medium
Describe polyacrylamide gel electrophoresis (PAGE)
- Electrophoresis is performed on a thin, vertical slab of polyacrylamide gel
- The gel acts as a molecular sieve
- The gel is inert
- All molecules are forced to move through the same matrix
- Smaller molecules move faster
Describe SDS-PAGE electrophoresis
- The proteins are first dissolved in SDS to disrupt non-covalent interactions
- Small proteins move rapidly through gel, large ones stay near top
mobility of proteins is linearly proportional to the log of their mass
Describe isoelectric focusing
A separation technique based on isoelectric points. Each protein moves until it reaches a position in the gel at which the pH = pI of the protein.
The gel gradient is formed by subjecting a mixture of polyampholytes (small multicharged polymers) which have many pI values to electrophoresis.
Describe two-dimensional electrophoresis
Isoelectric focusing is combined with SDS-PAGE to obtain very high resolution separations.
The proteins are subjected to IF and then horizontal SDS-PAGE.